Affect Cell Membrane Integrity Research Articles

Screening of the global health priority BoxⓇ reveals potential new disinfectants against the emerging multidrug-resistant pathogen Candida auris

BackgroundCandida auris has been identified by the World Health Organization as a critical pathogen due to its invasive nature, resistance to multiple drugs, and high mortality rates in hospital outbreaks. This fungus can persist on surfaces and human skin for extended periods, complicating infection control efforts. The need for effective disinfection strategies is urgent, as current disinfectants are often ineffective against C. auris biofilms. ObjectiveThe study aimed to identify potential disinfectants from a collection of 240 compounds in the Global Health Priority Box® that are effective against C. auris, particularly strains resistant to existing options. MethodsThe research employed a screening protocol using a fluconazole-resistant strain of C. auris (149/23). Antifungal activity was assessed using the microdilution method to determine Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs). Additional assays were conducted to evaluate biofilm inhibition, biofilm eradication, cell membrane integrity, nucleotide leakage, sorbitol protection assay, efflux pump inhibition, and hemolysis assay. ResultsTwo compounds, Hydramethylnon (MMV1577471) and Flufenerim (MMV1794206), demonstrated significant inhibitory effects against C. auris. Hydramethylnon exhibited potent antifungal activity, inhibiting up to 93 % of fungal growth with an MFC of 16 μg/mL. Flufenerim inhibited up to 58 % of fungal growth, showing fungistatic action with an MFC greater than 4 μg/mL. Biofilm inhibition tests showed that both compounds significantly inhibited biofilm formation, with increased efficacy at higher concentrations. Both compounds showed eradication rates in both stages. Furthermore, Hydramethylnon and Flufenerim did not affect cell membrane integrity or nucleotide leakage, suggesting a mode of action not reliant on disrupting these cellular components. The sorbitol protection assay revealed that neither compound caused cell wall damage. In the efflux pump inhibition assay, Hydramethylnon did not activate efflux pumps, while Flufenerim activated efflux pumps, reducing its effectiveness. Hemocompatibility assay showed safety. ConclusionThe study highlights Hydramethylnon and Flufenerim as promising candidates for further development as disinfectants, offering potential solutions to the urgent need for effective disinfection agents against C. auris. The findings underscore the value of screening compound collections to identify novel antifungal agents and understand their mechanisms of action, thereby contributing to the advancement of new disinfection strategies in healthcare settings.

Novel flavonoid derivatives containing 1,2,4-triazolo[4,3-a]pyridine as potential antifungal agents: Design, synthesis, and biological evaluation

A series of flavonoid derivatives containing 1,2,4-triazolo[4,3-a]pyridine were designed, synthesized and evaluated for their antifungal activity. Bioactivity tests showed that some target compounds exhibited the strong antifungal activity against Botrytis cinerea (B. cinerea), Sclerotinia sclerotiorum (S. sclerotiorum), and Phomopsis sp. Among them, the half maximal effective concentration (EC50) value of S2 against B. cinerea was 3.3 μg/mL, which was less than that of the control drug azoxystrobin (21.0 μg/mL). The EC50 value of S2 against S. sclerotiorum was 12.7 μg/mL, which was better than that of azoxystrobin (28.1 μg/mL). In addition, the in vivo protective and curative activities of S2 against blueberry were 92.0 and 79.8 % at 200 μg/mL, respectively, which were higher than those of the control drug azoxystrobin (89.4 and 71.7 %). Scanning electron microscopy (SEM) and spore germination experiments showed that S2 could not only cause the mycelium damage but also inhibited the spore germination. Fluorescence microscopy (FM) observation, relative electrical conductivity measurement and cytoplasmic leakage assays indicated that S2 could affect cell membrane integrity by inducing lipid peroxidation and increasing cell membrane permeability as well as causing leakage of cytoplasmic contents. These results show that flavonoid derivatives containing 1,2,4-triazolo[4,3-a]pyridine had excellent inhibitory effects on B. cinerea, providing another supplement for the development of new pesticides.

Polyvinyl Chloride Nanoparticles Affect Cell Membrane Integrity by Disturbing the Properties of the Multicomponent Lipid Bilayer in Arabidopsis thaliana.

The ubiquitous presence of nanoplastics (NPs) in natural ecosystems is a serious concern, as NPs are believed to threaten every life form on Earth. Micro- and nanoplastics enter living systems through multiple channels. Cell membranes function as the first barrier of entry to NPs, thus playing an important biological role. However, in-depth studies on the interactions of NPs with cell membranes have not been performed, and effective theoretical models of the underlying molecular details and physicochemical behaviors are lacking. In the present study, we investigated the uptake of polyvinyl chloride (PVC) nanoparticles by Arabidopsis thaliana root cells, which leads to cell membrane leakage and damage to membrane integrity. We performed all-atom molecular dynamics simulations to determine the effects of PVC NPs on the properties of the multicomponent lipid bilayer. These simulations revealed that PVCs easily permeate into model lipid membranes, resulting in significant changes to the membrane, including reduced density and changes in fluidity and membrane thickness. Our exploration of the interaction mechanisms between NPs and the cell membrane provided valuable insights into the effects of NPs on membrane structure and integrity.

Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver responses to chronic heat stress using DIA/SWATH

Aquaculture of rainbow trout (Oncorhynchus mykiss) is severely hampered by high temperatures in summer, and understanding the regulatory mechanisms controlling responses to chronic heat stress may assist the development of measures to relieve heat stress. In the present study, biochemical parameters revealed a strong stress response in rainbow trout at 24 °C, including activation of stress defence and immune systems. Liver proteome analysis under heat stress (24 °C) and control (18 °C) conditions using DIA/SWATH identified precursors (90,827), peptides (67,028), proteins (6770) and protein groups (5124), among which 460 differentially abundant proteins (DAPs; q-value < 0.05, fold change >1.5), 201 and 259 were up- and down-regulated, respectively. Many were related to heat shock proteins (HSPs), metabolism and immunity. Gene Ontology (GO) analysis showed that some DAPs induced at high temperature were involved in regulating cell homeostasis, metabolism, adaptive stress and stimulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified metabolic pathways, protein processing in endoplasmic reticulum, PPAR signalling, and complement and coagulation cascades. Protein-protein interaction (PPI) network analysis indicated that HSP90b1 and C3 may cooperative to affect cell membrane integrity under heat stress. Our findings assist the development of strategies to relieve heat stress in rainbow trout.

Revealing the astragalin mode of anticandidal action.

Due to limited arsenal of systemically available antifungal agents, infections caused by Candida albicans are difficult to treat and the emergence of drug-resistant strains present a major challenge to the clinicians worldwide. Hence further exploration of potential novel and effective antifungal drugs is required. In this study we have explored the potential of a flavonoid, astragalin, in controlling the growth of C. albicans, in both planktonic and biofilm forms by microdilution method; and in regulating the morphological switch between yeast and hyphal growth. Astragalin ability to interfere with membrane integrity, ergosterol synthesis and its role in the regulation of genes encoding for efflux pumps has been addressed. In our study, astragalin treatment produced good antimicrobial and significant antibiofilm activity. Anticandidal activity of astragalin was not related to ERG11 downregulation, neither to direct binding to CYP51 enzyme nor was linked to membrane ergosterol assembly. Instead, astragalin treatment resulted in reduced expression of CDR1 and also affected cell membrane integrity without causing cytotoxic effect on human gingival fibroblast cells. Considering that astragalin-mediated decreased expression of efflux pumps increases the concentration of antifungal drug inside the fungal cells, a combinatorial treatment with this agent could be explored as a novel therapeutic option for candidiasis.

Responses of Anammox Granular Sludge to Long-Term Rare Earth Element Feeding: Lanthanum as a Case

A tremendous input of ammonium and rare earth elements (REEs) has entered the surroundings on account of the discharge and leak of leaching agents during rare earth in-suit leaching mining, which has threatened various organisms. Anammox has the potential to release nitrogen contamination, but the potential impacts of REEs on anammox bacteria remain unclear. In this study, La (III) was chosen as a case to explore the long-term impacts on anammox granular sludge. The 5 mg L−1 La (III) which was examined hardly affected the anammox granulates because of the defense of extracellular polymeric substances. The high La concentrations (10–50 mg L−1) caused intercellular accumulation and the significant inhibition of nitrogen removal performance and dehydrogenase activity, especially a decrease in the relative abundance of Ca. Kuenenia. Moreover, it also induced patently oxidative damage and affected cell membrane integrity. Notably, extracellular polymeric substances have a limited defense capability; neither La3+ nor Ca2+/Mg2+ efflux-related genes aggravated the intracellular accumulation of La.

Effects of collected road dusts on cell viability, inflammatory response, and oxidative stress in cultured human corneal epithelial cells

Most studies on the adverse effects of air pollution have focused on respiratory and cardiovascular diseases, and there are relatively few studies on eye diseases following exposure of ambient particulate matter (PM). Epidemiological and clinical researches correlating the eye and PMs have recently received attention. PMs are complex mixture of particles that vary in chemical composition and size. This study investigated the influence of collected road dust on cell viability, inflammatory responses, and oxidative stress in human corneal epithelial cells. The collected road dust was classified with respect to aerodynamic diameter and solubility. Exposure concentration was calculated based on the particle deposition rate. We observed a dose-dependent decrease in cell viability at total PM2.5 and PM10. The pellet fractions of total PM2.5 and PM10 mainly contributed to the mitochondrial activity. Although both total PM2.5 and PM10 did not change the membrane integrity, the supernatant fractions significantly affected cell membrane integrity. Both total and fractions induced nitric oxide production and interleukin 8 expression. In addition, total PM2.5 and PM10 increased the oxidative stress; the pellet fractions of total PM2.5 and PM10 also induced higher oxidative stress. However, there was no significant difference between the cellular responses of total PM2.5 and PM10. We observed that the effects of collected road dust on cellular responses were strongly dependent on their concentration and solubility.

Innovative Self-Cleaning and Biocompatible Polyester Textiles Nano-Decorated with Fe-N-Doped Titanium Dioxide.

The development of innovative technologies to modify natural textiles holds an important impact for medical applications, including the prevention of contamination with microorganisms, particularly in the hospital environment. In our study, Fe and N co-doped TiO2 nanoparticles have been obtained via the hydrothermal route, at moderate temperature, followed by short thermal annealing at 400 °C. These particles were used to impregnate polyester (PES) materials which have been evaluated for their morphology, photocatalytic performance, antimicrobial activity against bacterial reference strains, and in vitro biocompatibility on human skin fibroblasts. Microscopic examination and quantitative assays have been used to evaluate the cellular morphology and viability, cell membrane integrity, and inflammatory response. All treated PES materials specifically inhibited the growth of Gram-negative bacilli strains after 15 min of contact, being particularly active against Pseudomonas aeruginosa. PES fabrics treated with photocatalysts did not affect cell membrane integrity nor induce inflammatory processes, proving good biocompatibility. These results demonstrate that the treatment of PES materials with TiO2-1% Fe–N particles could provide novel biocompatible fabrics with short term protection against microbial colonization, demonstrating their potential for the development of innovative textiles that could be used in biomedical applications for preventing patients’ accidental contamination with microorganisms from the hospital environment.

An Oleaginous Bacterium That Intrinsically Accumulates Long-Chain Free Fatty Acids in its Cytoplasm

Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

Isolation, characterization and mode of antimicrobial action againstVibrio choleraeof methyl gallate isolated fromAcacia farnesiana

The antimicrobial activity of Acacia farnesiana against Vibrio cholerae has been demonstrated; however, no information regarding its active compound or its mechanism of action has been documented. The active compound was isolated from A.farnesiana by bioassay-guided fractionation and identified as methyl gallate by nuclear magnetic resonance (NMR) techniques ((1) H NMR and (13) C NMR). The minimum bactericidal concentration (MBC) of methyl gallate and its effect on membrane integrity, cytoplasmic pH, membrane potential, ATP synthesis and gene expression of cholera toxin (ctx) from V.cholerae were determined. The MBC of methyl gallate ranged from 30±1 to 50±1μgml(-1) . Methyl gallate affected cell membrane integrity, causing a decrease in cytoplasmic pH (pHin , from 7·3 to <3·0), and membrane hyperpolarization, and ATP was no longer produced by the treated cells. However, methyl gallate did not affect ctx gene expression. Methyl gallate is a major antimicrobial compound from A.farnesiana that disturbs the membrane activity of V.cholerae. The effects of methyl gallate validate several traditional antimicrobial uses of A.farnesiana, and it is an attractive alternative to control V.cholerae.

Morphological and biochemical changes inPseudomonas fluorescensbiofilms induced by sub-inhibitory exposure to antimicrobial agents

Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of biomacromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 microg/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.

Cardiomyopathy Associated with Microcirculation Dysfunction in Laminin α4 Chain-deficient Mice

Laminin alpha4 chain is a component of extracellular matrix (ECM) laminin-8 and -9 and serves dual roles as a structure protein and as a signaling molecule. The abundance of laminin alpha4 chain transcripts in the heart suggests an important role of this protein in cardiovascular development and function. In this study, we demonstrate that laminin alpha4 deficient mice gradually develop cardiac hypertrophy with impaired function. We show that depletion of laminin alpha4 chain did not alter the levels of dystrophin-glycoprotein complex (DGC) components or affect cell membrane integrity. No alteration in integrin beta 1D protein was observed in terms of expression level or distribution pattern, indicating that the postnatal development of cardiac hypertrophy and cardiomyopathy in these mice is unlikely associated with the stability of sarcolemmal DGC and integrin complexes. Moreover, cardiomyocytes isolated from Lama4-/- mutant hearts maintained their contractility in vitro. In contrast, elevated levels of hypoxia-inducible factor 1alpha (Hif1alpha) and vascular endothelial growth factor A (Vegfa) transcripts, along with multiple foci of cardiomyocyte degeneration and fibrosis suggested sustained cardiac ischemia. Electron microscopy confirmed malformed blood vessels and wide pericapillary ECM spaces, suggesting the presence of microcirculation abnormalities in Lama4-/- mutant hearts. We thus conclude that mutation in the laminin alpha4 chain leads to abnormal cardiovascular ECM structure that cause insufficient oxygen supply to the heart and the subsequent ischemic cardiac phenotype observed. Our study links the genetic deficiency of an ECM protein to cardiomyopathy and implies a novel pathway of idiopathic cardiomyopathy in human.

Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides.

It is usually accepted that prion and amyloid-beta (A beta) peptides induce apoptotic cell death. However, the mechanisms that trigger neuronal death, induced by these amyloidogenic peptides, remain to be clarified. In the present study we analysed the neurotoxic effects of the synthetic prion and A beta peptides, PrP106-126 and A beta 25-35, in primary cultures of rat brain cortical cells. PrP106-126 and A beta 25-35 incubated at a concentration of 25 micro m for 24 h, did not affect cell membrane integrity, but decreased the metabolic capacity of the cells. The intracellular free Ca2+ concentration and reactive oxygen species levels increased significantly after 24 h treatment with PrP106-126 and A beta 25-35. Furthermore, these peptides (after 24 h exposure) also induced cytochrome c release from mitochondria and increased caspase-3-like activity. FK506, an inhibitor of the Ca2+/calmodulin-dependent phosphatase, calcineurin, was able to prevent cytochrome c release, caspase-3 activation and cell death induced by A beta 25-35 or PrP106-126 peptides. Taken together these data suggest that calcineurin is involved in A beta 25-35 and PrP106-126 neurotoxicity.

Hypoxia affects root sodium and chloride concentrations and alters water conductance in salt-treated jack pine (Pinus banksiana) seedlings

The effects of NaCl were studied in 6-month-old jack pine (Pinus banksiana Lamb.) seedlings growing in solution culture under hypoxic (approximately 2 mg l−1 O2) and well-aerated (approximately 8 mg l−1 O2) conditions. The results showed that hypoxia led to further reduction of stomatal conductance (g s) in plants treated with 45 mM NaCl. This effect was likely due to a reduction in root hydraulic conductance by both stresses. When applied individually or together, neither 45 mM NaCl nor hypoxia affected cell membrane integrity of needles as measured by tissue electrolyte leakage. Hypoxia did not alter shoot Na+ and Cl− concentrations in NaCl-treated plants. However, root Na+ concentrations were lower in NaCl-treated hypoxic plants, suggesting that hypoxia affected the ability of roots to store Na+. Hypoxia also induced root electrolyte leakage from NaCl-treated and control plants. The higher root Cl− concentrations compared with Na+ and the positive correlation between root Cl− concentrations and electrolyte leakage suggest that Cl− played a major role in salt injury observed in jack pine seedlings. Roots of well-aerated plants treated for 1 week with NaCl contained almost two-fold higher concentration of total non-structural carbohydrates compared with plants from other experimental treatments and these concentrations decreased in subsequent weeks. We suggest that under prolonged hypoxic conditions, roots lose the ability to prevent Cl− uptake resulting in the increase in root Cl− concentration, which has damaging effects on root cell membranes.

Involvement of oxidative stress in the enhancement of acetylcholinesterase activity induced by amyloid beta-peptide

Acetylcholinesterase (AChE) activity is increased within and around amyloid plaques, which are present in Alzheimer's disease (AD) patient's brain. In this study, using cultured retinal cells as a neuronal model, we analyzed the effect of the synthetic peptide Aβ 25–35 on the activity of AChE, the degradation enzyme of acetylcholine, as well as the involvement of oxidative stress in this process. The activity of AChE was increased when retinal cells were incubated with Aβ 25–35 (25 μM, 24 h) and antioxidants such as α-tocopherol acetate and nitric oxide synthase (NOS) inhibitors were capable of preventing this effect. Despite Aβ 25–35 did not affect cell membrane integrity, the redox capacity of cells decreased. The incubation with this amyloidogenic peptide led to an increment of reactive oxygen species formation (20%), of lipid peroxidation (65%), and basal intracellular calcium levels (40%). The data obtained show that the enhancement of AChE activity induced by Aβ 25–35 is mediated by oxidative stress, and that vitamin E and NOS inhibitors, by preventing the compromise of the enzyme activity, can have an important role in the maintenance of acetylcholine synaptic levels, thus preventing or improving cognitive and memory functions of AD patients.

Induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor RPR-115135: role of gene mutations.

To investigate the relationship between oncogene activation and induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor, RPR-115135, two isogenic cell lines, human colon cancer line HCT-116, which harbors a K-ras mutation, and spontaneously immortalized human breast epithelial cell line MCF-10A, were utilized. HCT-116 cells were transfected with an empty control pCMV vector (clone CMV-2) or with a dominant negative mutated p53 transgene (clone Mu-p53-2) to disrupt p53 function. In both clones RPR-115135 induced a significant increase in the frequency of micronucleation at concentrations that did not affect cell membrane integrity. RPR-115135 produced a significant increase in the ratio of CREST+ to CREST- micronuclei. MCF-10A cells were stably transfected with either c-Ha-ras or c-erbB-2 or both H-ras + c-erbB-2. No induction of micronuclei was observed. No induction of micronuclei was reported in human lymphocytes and in primary spinal cells obtained from 7-day chick embryos. In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.

Modulation of APP processing and secretion by okadaic acid in primary guinea pig neurons.

Primary cultures of guinea pig neurons were used as a model system to study the influence of the protein phosphatase inhibitor okadaic acid (OA) on the secretion, processing and phosphorylation of the amyloid precursor protein (APP). This primary cell culture system mimics more closely than other cell culture systems the human in vivo condition, as guinea pig APP is 98% homologous to human APP at the protein level, identical regarding the Abeta sequence and is processed in a similar manner as human APP. Both intracellular and secreted APP was upregulated by OA treatment (0.3 nM-10 nM) of 14 days old cultures in a concentration dependent manner while the amount of Abeta in the medium was decreased. OA treatment did not affect cell membrane integrity of primary neurons but induced DNA fragmentation. Phosphorylation of APP was unchanged by the low OA concentration used. These results show that OA treatment of guinea pig primary cultures might be used as a model to study the effects of modulation of signal transduction on secretion and processing of APP.

Triglyceride disposition in isolated hepatocytes after treatment with hydrazine

Treatment of animals with hydrazine causes the accumulation of triglycerides in the liver but the mechanism remains unclear. Therefore, the effect of hydrazine on hepatic triglyceride synthesis and subsequent transport was studied in a hepatocyte model, in vitro in order to isolate liver cells from extrahepatic influences. Hepatocytes were isolated and either incubated in suspension with [ 14C]palmitate in the presence of hydrazine (2–12 mM) or pre-incubated with [ 14C]palmitate, washed free of the fatty acid and then incubated with hydrazine (2–12 mM). Hydrazine resulted in a significant reduction in the incorporation of [ 14C]palmitate into triglycerides and reduction in the transportation of triglycerides out of cells. When [ 14C]palmitate was in the incubation medium, ATP levels were reduced by lower concentrations of hydrazine than have previously been reported. None of the concentrations of hydrazine used affected cell membrane integrity (viability) as measured by LDH leakage. The 14CO 2 produced by the β-oxidation of [ 14C]palmitate was also measured in short term incubations (30 min) carried out in sealed vessels. There was a dose dependent increase in 14CO 2 produced by very low concentrations of hydrazine (0.01–0.1 mM) after which the effect was maximal and concentrations above 8 mM hydrazine decreased 14CO 2 production. The data suggest that the inhibition of transportation of triglycerides out of cells by hydrazine may have a more important role in the accumulation of triglycerides in the liver than has been previously recognised. However, the model was not able to mimic the accumulation of triglycerides in hepatocytes seen in vivo.

Primary cultured normal human hepatocytes for hepatitis B virus receptor studies

Background/Aims: We analyzed the hepatitis B virus envelope specificities (HBs, preS2 and preS1) involved in virus attachment to normal human hepatocytes, and we performed in vitro hepatitis B virus infection experiments without addition of dimethyl sulfoxide and polyethylene glycol, which may affect cell membrane integrity, in order to study further the early steps of the life cycle of the hepatitis B virus. Methods: Primary normal human hepatocytes were prepared from surgical biopsies by the two-step collagenase perfusion technique, and cultured in a fetal calf serum-free medium supplemented with 10 −6 M dexamethasone. Cell-binding assays, ligand blotting and immunohistochemistry experiments were carried out using our anti-idiotypic (Ab2) antibodies (Ab2s/preS1, Ab2s/preS2 and Ab2s/HBs). Results: Probing primary normal human hepatocytes, the 35-kDa major preS1-binding protein (preS1-BP35) we have previously identified in human hepatoma HepG2 cells was recognized in blotting, whereas both HBs- and preS1-specificities of the hepatitis B virus envelope interacted strongly with normal human hepatocyte cell membrane in cell-binding assays and immunohistochemistry experiments. Hepatitis B virus infectivity studies confirmed a great inter-experimental variability depending on donors and liver perfusion, and demonstrated a great intra-experimental variability depending on the serum-derived hepatitis B virus isolate used for the inoculation. In our culture conditions, only increased detection of the RC and CCC DNA forms of hepatitis B virus in cells and of hepatitis B virus surface antigens in medium was observed 4 to 8 days after exposure of cells to hepatitis B virus. Conclusion: These findings support a potential role for preS1-BP35 as a receptor protein for hepatitis B virus. In our hands, limitation(s) in the hepatitis B virus life cycle may occur at some step after virion binding, and likely result from complex regulation of reverse transcription of the RNA and translation of core protein by extrahepatic host factors or/and by the virus itself. However, the normal human hepatocyte model developed here is available for studying the initial steps in hepatitis B virus entry into cells.

Effect of cadmium and nickel on ethylene biosynthesis in soybean

Exogenously supplied cadmium and nickel considerably affected the ethylene biosynthesis in soybean cuttings. Cadmium stimulated ethylene production by increasing production of free ACC and stimulating EFE activity. Nickel inhibited ethylene production by depressing EFE activity, but stimulated the production of free ACC. Both heavy metals did not apparently affect cell membrane integrity.